Mutants of streptococcal toxin c and methods of use

ABSTRACT

This invention is directed to mutant SPE-C toxins or fragments thereof, vaccine and pharmaceutical compositions, and methods of using the vaccine and pharmaceutical compositions. The preferred SPE-C toxin has at least one amino acid change and is substantially non-lethal compared with the wild type SPE-C toxin. The mutant SPE-C toxins can form vaccine compositions useful to protect animals against the biological activities of wild type SPE-C toxin.

BACKGROUND OF THE INVENTION

[0001]Streptococcus pyogenes, also known as β-hemolytic group Astreptococci (GAS) is a pathogen of humans which can cause mildinfections such as pharyngitis and impetigo. Post infection autoimmunecomplications can occur, namely rheumatic fever and acuteglomerulonephritis. GAS also causes severe acute diseases such asscarlet fever and streptococcal toxic shock syndrome (STSS). Severe GASinfections were a large problem in the U.S. and throughout the world atthe beginning of this century. In the mid-forties, the number of casesand their severity decreased steadily for reasons not yet completelyunderstood. However, more recently, a resurgence of serious diseasescaused by GAS has been seen such that there may be 10-20,000 cases ofSTSS each year in the United States. As many as 50 to 60% of thesepatients will have necrotizing fascitis and myositis; 30 to 60% will dieand as many as one-half of the survivors will have limbs amputated.

[0002] In 1986 and 1987 two reports described an outbreak of severe GASinfections localized in the Rocky Mountain area. These reports have beenfollowed in the past few years by several others describing a diseasewith analogous clinical presentation. The symptoms described for thisdisease were very similar to those described for toxic shock syndrome(TSS), and in 1992 a committee of scientists gave to this clinicalpresentation the formal name of STSS, and set the criteria for itsdiagnosis. STSS is defined by the presence of the following:

[0003] 1. hypotension and shock;

[0004] 2. isolation of group A streptococci;

[0005] 3. two or more of the following symptoms: fever 38.5° C. orhigher, scarlet fever rash, vomiting and diarrhea, liver and renaldysfunction, adult respiratory distress syndrome, diffuse intravascularcoagulation, necrotizing fascitis and/or myositis, bacteremia.

[0006] Streptococcal isolates from STSS patients are predominantly of Mtype 1 and 3, with M18 and nontypable organisms making up most of thereminder. The majority of M1, 3, 18, and nontypable organisms associatedwith STSS make pyrogenic exotoxin type A (approx. 75%) with theremainder of the isolates making pyrogenic exotoxin type C (SPE-C).Moreover, administration of SPE-C to a rabbit animal model and inaccidental human inoculations can reproduce the symptoms of STSS. Inaddition to SPE-C association with STSS studies have shown that group Astreptococcal isolates from rheumatic fever and guttate psoriasispatients make SPE-C.

[0007] SPE-C is a single peptide of molecular weight equal to 24,000daltons. speC, the gene for SPE-C, has been successfully cloned andexpressed in Escherichia coli. SPE-C is a member of a large family ofexotoxins produced by streptococci and staphylococci, referred to aspyrogenic toxins based upon their ability to induce fever and enhancehost susceptibility up to 100,000 fold to endotoxin.

[0008] Recently these toxins have been referred to as superantigensbecause of their ability to induce massive proliferation of Tlymphocytes, regardless of their antigenic specificity, and in a fashiondependent on the composition of the variable part of the β chain of theT cell receptor. These toxins also stimulate massive release of IFN-γ,IL-1, TNF-α and TNF-β. Other members of this family are streptococcalpyrogenic exotoxins type A and B, staphylococcal toxic shock syndrometoxin 1, staphylococcal enterotoxins A, B, Cn, D, E, G and H, andnon-group A streptococcal pyrogenic exotoxins. These toxins have similarbiochemical properties, biological activities and various degrees ofsequence similarity.

[0009] The most severe manifestations of STSS are hypotension and shock,that lead to death. It is generally believed that leakage of fluid fromthe intravascular to the interstitial space is the final cause ofhypotension, supported by the observation that fluid replacement therapyis successful in preventing shock in the rabbit model of STSS describedabove. It has been hypothesized that SPE-C may act in several ways onthe host to induce this pathology.

[0010] SPE-C has been shown to block liver clearance of endotoxin ofendogenous flora's origin, by compromising the activity of liver Kuppfercells. This appears to cause a significant increase in circulatingendotoxin, that through binding to lipopolysaccharide binding protein(LBP) and CD14 signaling leads to macrophage activation with subsequentrelease of TNF-α and other cytokines. Support for the role of endotoxinin the disease is given by the finding that the lethal effects of SPE-Ccan be at least partially neutralized by the administration to animalsof polymyxin B or by the use of pathogen free rabbits.

[0011] Another modality of induction of shock could be the directactivity of the toxin on capillary endothelial cells. This hypothesisstems from the finding that the staphylococcal pyrogenic toxin TSST-1binds directly to human umbilical cord vein cells and is cytotoxic toisolated porcine aortic endothelial cells.

[0012] Another of the toxin's modality of action includes itssuperantigenicity, in which the toxin interacts with and activates up to50% of the host T lymphocytes. This massive T cell stimulation resultsin an abnormally high level of circulating cytokines TNF-β and IFN-γwhich have direct effects on macrophages to induce release of TNF-α andIL-1. These cytokines may also be induced directly from macrophages bySPE-C through MHC class II binding and signaling in the absence of Tcells. The elevated levels of TNF-α and -β cause several effectstypically found in Gram negative induced shock, among which is damage toendothelial cells and capillary leak. However, the administration toSPE-A treated rabbits of cyclosporin A, which blocks upregulation ofIL-2 and T cell proliferation, did not protect the animals from shock,suggesting that additional mechanisms may be more important in causingcapillary leak.

[0013] Thus, there is a need to localize sites on the SPE-C moleculeresponsible for different biological activities. There is a need todevelop variants of SPE-C that have changes in biological activitiessuch as toxicity and mitogenicity. There is a need to developcompositions useful in vaccines to prevent or ameliorate streptococcaltoxic shock syndrome. There is also a need to develop therapeutic agentsuseful in the treatment of streptococcal toxic shock syndrome and otherdiseases.

SUMMARY OF THE INVENTION

[0014] This invention includes mutant SPE-C toxins and fragmentsthereof, vaccines and pharmaceutical compositions and methods of usingvaccines and pharmaceutical compositions.

[0015] Mutant SPE-C toxins have at least one amino acid change and aresubstantially nonlethal as compared with a protein substantiallycorresponding to a wild type SPE-C toxin. For vaccine compositions,mutant toxins also stimulate a protective immune response to at leastone biological activity of a wild type SPE-C toxin. Mutant toxins forvaccine compositions are optionally further selected to have a decreasein enhancement of endotoxin shock and a decrease in T cell mitogenicitywhen compared to the wild type SPE-C. For pharmaceutical compositions,it is preferred that a mutant toxin is substantially nonlethal whilemaintaining T cell mitogenicity comparable to a wild type SPE-C toxin.

[0016] The invention also includes fragments of a wild type SPE-C toxinand mutants of SPE-C toxins. Fragments and peptides derived from wildtype SPE-C are mutant SPE-C toxins. Fragments can include differentdomains or regions of the molecule joined together. A fragment issubstantially nonlethal when compared to a wild type SPE-C toxin. Formutant toxins, a fragment has at least one amino acid change compared toa wild type SPE-C amino acid sequence. Fragments are also useful invaccine and pharmaceutical compositions.

[0017] The invention also includes expression cassettes, vectors andtransformed cells. An expression cassette comprises a DNA sequenceencoding a mutant SPE-C toxin or fragment thereof operably linked to apromoter functional in a host cell. DNA cassettes are preferablyinserted into a vector. Vectors include plasmids or viruses. Vectors areuseful to provide template DNA to generate DNA encoding a mutant SPE-Ctoxin. DNA cassettes and vectors are also useful in vaccinecompositions. Nucleic acids encoding a mutant SPE-C toxin or fragmentthereof can be delivered directly for expression in mammalian cells. Thepromoter is preferably a promoter functional in a mammalian cell.Nucleic acids delivered directly to cells can provide for expression ofthe mutant SPE-C toxin in an individual so that a protective immuneresponse can be generated to at least one biological activity of a wildtype SPE-C toxin.

[0018] Additional vaccine compositions include stably transformed cellsor viral vectors including an expression cassette encoding a mutantSPE-C toxin or fragment thereof. Viral vectors such as vaccinia can beused to immunize humans to generate a protective immune response againstat least one biological activity of a wild type SPE-C toxin. Transformedcells are preferably microorganisms such as S. aureus, E. coli, orSalmonella species spp. Transformed microorganisms either include mutantSPE-C toxin or fragment thereof on their surface or are capable ofsecreting the mutant toxin. Transformed microorganisms can beadministered as live, attenuated or heat killed vaccines.

[0019] The invention also includes methods of using vaccines andpharmaceutical compositions. Vaccines are administered to an animal inan amount effective to generate a protective immune response to at leastone biological activity of a wild type SPE-C toxin. Preferably, thevaccine compositions are administered to humans and protect against thedevelopment of STSS. Pharmaceutical compositions are used in methods ofstimulating T cell proliferation.

[0020] The mutant SPE-C toxins and/or fragments thereof and othervaccine compositions can be useful to generate a passive immune serum.Mutant SPE-C toxins or fragments thereof, DNA expression cassettes orvectors, or transformed microorganisms can be used to immunize an animalto produce neutralizing antibodies to at least one biological activityof wild type SPE-C. The neutralizing antibodies immunoreact with amutant SPE-C toxin and/or fragment thereof and the wild type SPE-Ctoxin. Passive immune serum can be administered to an animal withsymptoms of A streptococcal infection and STSS.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIG. 1 shows the nucleotide sequence of speC. Numbering is inreference to the ATG start codon. Possible promoter (−10, −35) andShine-Dalgarno (SD) sequences are noted. The deduced amino acid sequenceis given below the nucleotide sequence. An asterisk after residue 27indicates the cleavage site between the signal peptide and matureprotein. Overlined nucleotides 3′ of the translation stop codon arepalindromic sequences.

[0022]FIG. 2 shows a front view of a ribbon structure of SPE-C.

[0023]FIG. 3 shows a back view of a ribbon structure of SPE-C.

[0024]FIG. 4 shows a front view of a ribbon structure of SPE-C orientedto show locations contacting major histocompatibility complex type II ina complex.

[0025]FIG. 5 shows a front view of a ribbon diagram of SPE-C oriented toshow locations that contact the T cell receptor in a complex.

[0026]FIG. 6 shows a rear view of a ribbon structure of SPE-C orientedto show residues of the central α helix that form the floor of thegroove that contacts the liver renal tubular cell receptor in a complexwith this receptor.

[0027]FIG. 7 shows mitogenic activity of single mutants Y15A and N38A.

[0028]FIG. 8 shows mitogenic activity of single mutants Y17A.

[0029]FIG. 9 shows mitogenic activity of double mutants Y15A/N38A andY17A/N38A.

[0030]FIG. 10 shows front and back views of a ribbon structure of SPE-Cshowing residues substituted in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

[0031] This invention is directed to mutant SPE-C toxins and fragmentsthereof, vaccine and pharmaceutical compositions including mutant SPE-Ctoxins or fragments thereof, methods of preparing mutant SPE-C toxinsand fragments thereof, and methods of using SPE-C toxins and fragmentsthereof.

[0032] Mutant SPE-C toxins are proteins that have at least one aminoacid change and have at least one change in a biological functioncompared with a protein substantially corresponding to a wild type SPE-Ctoxin. Preferably, the mutant SPE-C toxin is substantially nonlethalwhen compared to a wild type SPE-C toxin at the same dose. Mutant SPE-Ctoxins can be generated using a variety of methods includingsite-directed mutagenesis, random mutagenesis, conventional mutagenesis,in vitro mutagenesis, spontaneous mutagenesis and chemical synthesis.Mutant SPE-C toxins are preferably selected to: 1) ensure at least onechange in an amino acid; and 2) to have a change in at least onebiological function of the molecule preferably a decrease or eliminationof systemic lethality. The mutant toxins are useful in vaccinecompositions for protection against at least one biological activity ofSPE-C toxin such as prevention or amelioration of STSS and in methods oftreating animals with symptoms of STSS.

[0033] A. Mutant SPE-C Toxins or Fragments Thereof, Vaccine andPharmaceutical Compositions

[0034] The invention includes mutant SPE-C toxins that have at least oneamino acid change and that have at least one change in a biologicalactivity compared with proteins that substantially correspond to andhave the same biological activities as wild type SPE-C.

[0035] Wild type SPE-C toxin is encoded by a gene speC. The wild typeSPE-C toxin has a molecular weight of 24,000 Daltons as determined bySDS PAGE of purified protein. A DNA sequence encoding a wild type SPE-Ctoxin and the predicted amino acid sequence for a wild type SPE-C toxinis shown in FIG. 1. A DNA sequence encoding a wild type SPE-A toxin hasbeen cloned in E. coli and S. aureus. Amino acid number designations inthis application are made by reference to the sequence of FIG. 1 withaspartate at position 28 designated as the first amino acid. The first27 amino acids represent a leader sequence not present in the matureprotein.

[0036] The wild type SPE-C toxin has several biological activities.These biological activities include: 1) fever; 2) STSS; 3) systemiclethality due to development of STSS or enhancement of endotoxin shock;4) enhancing endotoxin shock; 5) induction of capillary leak andhypotension; 6) inducing release of cytokines such as IFN γ, IL-1, TNF-αand TNF-β; 7) binding to porcine aortic endothelial cells; 8) binding toMHC class II molecules; 9) binding to T-cell receptors; and 10) T-cellmitogenicity (superantigenicity). These activities can be assayed andcharacterized by methods known to those of skill in the art.

[0037] As used herein, the definition of a wild type SPE-C toxinincludes variants, such as allelic variants, of a wild type SPE-C toxinthat have the same biological activity of wild type SPE-C toxin. TheseSPE-C toxins may have a different amino acid or their genes may have adifferent nucleotide sequence from that shown in FIG. 1 but do not havedifferent biological activities. Changes in amino acid sequence arephenotypically silent. Preferably, these toxin molecules have systemiclethality and enhance endotoxin shock to the same degree as wild typeSPE-C toxin shown in FIG. 1. Preferably these toxins have at least60-99% homology with wild type SPE-C toxin amino acid sequence as shownin FIG. 1 as determined using the SS2 Alignment Algorithm as describedby Altschul,S. F., Bull. Math. Bio. 48:603 (1986). Proteins that havethese characteristics substantially correspond to a wild type SPE C.

[0038] A mutant SPE-C toxin is a toxin that has at least one change in aamino acid compared with a protein substantially corresponding to a wildtype SPE-C toxin. The change can be an amino acid substitution,deletion, or addition. There can be more than one change in the aminoacid sequence, preferably 1 to 6 changes. It is preferred that there ismore than one change in the amino acid sequence to minimize thereversion of mutant SPE-C toxin to the wild type SPE-C toxin havingsystemic lethality or toxicity. For mutant SPE-C toxins useful invaccines, it is preferred that the change in the amino acid sequence ofthe toxin does not result in a change of the toxin's ability tostimulate an antibody response that can neutralize wild type SPE-Ctoxin. For mutant SPE-C toxins useful in vaccines, it is especiallypreferred that the mutant toxins are recognized by polyclonalneutralizing antibodies to SPE-C toxin such as from Toxin Technologiesin Boca Raton, Fla. or Dr. Schlievert (University of Minnesota,Minneapolis, Minn.) and that the proteolytic profile is not alteredcompared with wild type SPE-C.

[0039] The changes in the amino acid sequence can be site-specificchanges at one or more selected amino acid residues of a wild type SPE-Ctoxin. Site-specific changes are selected by identifying residues inparticular domains of the molecule as described or at locations wherecysteine residues are located. Site-specific changes at a particularlocation can optionally be further selected by determining whether anamino acid at a location or within a domain is identical to or hassimilar properties to an equivalent residue in other homologousmolecules by comparison of primary sequence homology or 3-Dconformation. A homologous molecule is one that can be identified bycomparison of primary sequence homology using the SS2 alignmentalgorithm of Altschul et al., cited supra or a homology modeling programsuch as Insight/Homology from BioSym, San Diego, Calif. A homologousmolecule is one that displays a significant number, typically 30-99%, ofidentical or conservatively changed amino acids or has a similar threedimensional structure, typically RMS error for conserved regions of <2Angstroms, when compared to a wild type SPE-C toxin.

[0040] Changes in the amino acid sequence at a particular site can berandomly made or specific changes can be selected. Once a specific siteis selected it is referred to by its amino acid number designation andby the amino acid found at that site in the wild type SPE-C as shown inFIG. 1. The amino acid number designations made in this application areby reference to the sequence in FIG. 1 with the aspartate at position 28being counted as the first amino acid. Equivalent amino acidscorresponding to those identified at a particular site in proteinssubstantially corresponding to a wild type SPE-C toxin may havedifferent amino acid numbers depending on the reference sequence or ifthey are fragments. Equivalent residues are also those found inhomologous molecules that can be identified as equivalent to amino acidsin proteins substantially corresponding to a wild type SPE-C toxineither by comparison of primary amino acid structure or by comparison toa modeled structure as shown in FIG. 1 or by comparison to a knowncrystal structure of a homologous molecule. It is intended that theinvention cover changes to equivalent amino acids at the same or similarlocations regardless of their amino acid number designation.

[0041] If a specific substitution is selected for an amino acid at aspecific site, the amino acid to be substituted at that location isselected to include a structural change that can affect biologicalactivity compared with the amino acid at that location in the wild typeSPE-C. The substitution may be conservative or nonconservative.Substitutions that result in a structural change that can affectbiological activity include: 1) change from one type of charge toanother; 2) change from charge to noncharged; 3) change in cysteineresidues and formation of disulfide bonds; 4) change from hydrophobic tohydrophilic residues or hydrophilic to hydrophobic residues; 5) changein size of the amino acids; 6) change to a conformationally restrictiveamino acid or analog; and 7) change to a non-naturally occurring aminoacid or analog. The specific substitution selected may also depend onthe location of the site selected. For example, it is preferred thatamino acids in the N-terminal alpha helix have hydroxyl groups tointeract with exposed amide nitrogens or that they be negatively chargedto interact with the partial positive charge present at the N-terminusof the a helix.

[0042] Mutant toxins may also include random mutations targeted to aspecific site or sites. Once a site is selected, mutants can begenerated having each of the other 19 amino acids substituted at thatsite using methods such as described by Aiyar et al., Biotechniques14:366 (1993) or Ho et al. Gene 77:51-54 (1984). In vitro mutagenesiscan also be utilized to substitute each one of the other 19 amino acidsor non-naturally occurring amino acids or analogs at a particularlocation using a method such as described by Anthony-Cahill et al.,Trends Biochem. Sci. 14:400 (1989).

[0043] Mutant toxins also include toxins that have changes at one ormore sites of the molecule not specifically selected and that have achange in amino acids that is also not specifically selected but can beany one of the other 19 amino acids or a non-naturally occurring aminoacid.

[0044] Substitutions at a specific site can also include but are notlimited to substitutions with non-naturally occurring amino acids suchas 3-hydroxyproline, 4-hydroxyproline, homocysteine, 2-aminoadipic acid,2-aminopimilic acid, ornithine, homoarginine, N-methyllysine, dimethyllysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4-diaminobutryicacid, hydroxylysine, substituted phenylalanine, norleucine, norvaline,(-valine and halogenated tyrosines. Substitutions at a specific site canalso include the use of analogs which use non-peptide chemistryincluding but not limited to ester, ether and phosphoryl and boronlinkages.

[0045] The mutant toxins can be generated using a variety of methods.Those methods include site-specific mutagenesis, mutagenesis methodsusing chemicals such as EMS, or sodium bisulfite or UV irradiation, byspontaneous mutation, by in vitro mutagenesis and chemical synthesis.Methods of mutagenesis can be found in Sambrook et al., A Guide toMolecular Cloning, Cold Spring Harvard, N.Y. (1989). The especiallypreferred method for site-specific mutagenesis is using asymmetric PCRwith three primers as described by Perrin and Gilliland, 1990. NucleicAcid Res. 18:7433.

[0046] Superpositioning the three-dimensional structures of fourstaphylococcal superantigens (TSST-1, SEA, SEB, and SEC-3) and of SPE-Cdemonstrated that these proteins share 16 structurally conserved aminoacids (Table 1). Using these 16 structurally conserved amino acidresidues as reference points allows superpositioning of the structuresof these 5 proteins with RMS (root mean square) differences at or below2 angstroms, which is significant for proteins with minimal amino acidsequence conservation. This superpositioning based on 16 structurallyconserved amino acids allows detailed comparison of the structure ofSPE-C with the staphylococcal superantigens.

[0047] The crystal structure of the complex of staphylococcalsuperantigen SEB and the class II major histocompatibility complex(MHC-II) shows amino acids on SEB that contact MHC-II, and includesthose residues listed in Table 2. Superposition of the SPE-C structureindicates the location of portions of SPE-C that contact MHC-II in acomplex of these two proteins. These locations are shown in FIG. 4 asballs.

[0048] Specifically, with reference to FIG. 4, these include locations 1and 2 on strand 3 of β-barrel 4 of B-subunit 5. Location 1 is theposition of an amino acid 1 residue past a type 4 turn or bulge instrand 3 and about three residues from the junction of strand 3 and loop6. Location 1 can be occupied by a polar amino acid preferably Thr-33 ofSPE-C. Location 2 represents the amino acid in strand 3 closest to thejunction of strand 3 and loop 6, while remaining on the strand. Location2 can be occupied by a polar amino acid, preferably His-35 of SPE-C.Location 7 represents the amino acid nearest the junction of strand 3and loop 6. Loop 6 is a “type 1” or “type 2” turn. Location 7 can be ahydrophobic amino acid, preferably Leu-36 of SPE-C. β -Barrel 4 ofB-subunit 5 also includes residue Asn-38.

[0049] Location 8 is in loop 6. Location 8 can be occupied by a polaramino acid, preferably Asn-37 of SPE-C. Locations 10, 11 and 14 are onstrand 12. Location 10 is the amino acid on strand 12 nearest thejunction with loop 13. Location 10 can be a charged amino acid,preferably Arg-44 of SPE-C. Location 11 is at about the middle of strand12 and can be occupied by a charged amino acid, preferably Lys-42 ofSPE-C. Location 14 is at the junction of strand 12 and loop 6 but onstrand 12. Location 14 can be occupied by a polar amino acid, preferablyThr-40 of SPE-C. Location 15 is on loop 6. Location 15 can be occupiedby a charged amino acid, preferably Asp-39 of SPE-C.

[0050] Locations 17-20 are on strand 21. Locations 19 and 20 areadjacent. Locations 17-19 are separated with room enough for a locationbetween each. Location 17 is about one amino acid from the junction ofstrand 21 and loop 13. Location 17 can be occupied by a hydrophobicamino acid, preferably Ile-50 of SPE-C. Location 18 is at approximatelythe midpoint of strand 21 and can be occupied by a neutral or polaramino acid, preferably amino acid Ser-52 of SPE-C. Location 19 is abouttwo amino acids from the junction of strand 21 and loop 22. Location 19can be occupied by a neutral or polar amino acid, preferably Met-54 ofSPE-C. Location 20 is in strand 21 adjacent to the junction of strand 21with loop 22. Location 20 can be occupied by a neutral or polar aminoacid, preferably Ser-55 of SPE-C. Location 23 is on alpha helix 24 inthe turn and ending with the junction of helix 24 and loop 25. Location23 is on a face of helix 24 facing location 20. Location 23 can beoccupied by a neutral amino acid, preferably Ala-186 of SPE-C.

[0051] The crystal structure of the complex of staphylococcalsuperantigen SEC-3 and the T cell receptor shows amino acids on SEC-3that contact T cell receptor, and includes residues listed in Table 3.Superposition of the SPE-C structure indicates the location of aminoacids of SPE-C that contact the T cell receptor in a complex of thesetwo proteins. These locations are shown in FIG. 5 as balls.

[0052] Specifically, with reference to FIG. 5, these include location 26which is at the junction of loop 13 and strand 21. Location 26 can beoccupied by a polar amino acid, preferably Tyr-49 of SPE-C. Location 27is on strand 28 approximately the distance of three amino acids from thejunction of strand 28 with loop 29. Location 27 can be occupied by apolar amino acid, preferably Tyr-85 of SPE-C. Location 30 is on loop 29approximately equidistant between strands 28 and 32. Location 30 can beoccupied by a polar amino acid, preferably His-81 of SPE-C. Location 31is at the junction of loop 29 and strand 32. Location 31 can be occupiedby a polar amino acid, preferably Asn-79 of SPE-C. Locations 33-36 areon strand 32. Location 33 is the amino acid adjacent to the junction ofstrand 32 and loop 31. Position 33 can be occupied by hydrophobic aminoacid, preferably Leu-78 of SPE-C. Location 34 is one amino acid fromlocation 33. Location 34 can be occupied by a hydrophobic amino acid,preferably Ile-77 of SPE-C. Location 35 can be occupied by a polar aminoacid, preferably Tyr-76 of SPE-C. Location 36 can be occupied by ahydrophobic amino acid, preferably Phe-75 of SPE-C.

[0053] Location 38 is a residue on irregular alpha helix 24 on a turn ofthat alpha helix nearest the junction with loop 39. Location 38 is on aportion of the turn nearest strand 40. Location 38 can be occupied by acharged amino acid, preferably Asp-183 of SPE-C. Location 41 is on loop39 approximately one amino acid from the junction of loop 39 and alphahelix 24. A side chain on an amino acid at location 41 is orientedtoward central alpha helix 42. Location 41 can be occupied by a chargedamino acid, preferably Arg-181 of SPE-C.

[0054] Loop 43 includes locations 44, 45, 46, and 47. Locations 44-47are adjacent locations on the portion of loop 43 most exposed to thesolvent. Location 44 can be occupied by a charged amino acid, preferablyGlu-178 of SPE-C. Location 45 can be occupied by a polar amino acid,preferably Tyr-153 of SPE-C. Location 46 can be occupied by a chargedamino acid, preferably Asp-148 of SPE-C. Location 47 can be occupied bya polar amino acid, preferably Tyr-147 of SPE-C.

[0055] Locations 48-50 are on N-terminal alpha helix 51. Locations 48and 49 are on a turn of alpha helix 51 nearest the junction with loop52. Location 48 can be occupied by a neutral or polar amino acid,preferably Ser-11 of SPE-C. Location 49 can be occupied by a chargedamino acid, preferably Asp-12 of SPE-C. Locations 48 and 49 representadjacent amino acid positions. Location 50 is in the turn of alpha helix51 adjacent to the junction with loop 53. Location 50 is on the portionof that turn that is most solvent-exposed. Location 50 can be occupiedby a polar amino acid, preferably Tyr-15 of SPE-C. N-terminal alphahelix 51 also includes residue Tyr-17.

[0056] SPE-C binds a liver renal tubular cell receptor at a siteincluding residues on a groove on the “back” of SPE-C. Locations 54-60define a surface of a groove on SPE-C between B subunit 5 and A subunit61 that is part of the interaction with the liver renal tubular cellreceptor. Locations 54-59 are on central alpha helix 42. Location 60 ison loop 16 adjacent to the junction of loop 16 with central alpha helix42. Location 54 can be occupied by a polar amino acid, preferablyAsn-143 of SPE-C. Location 55 can be occupied by a charged amino acid,preferably Asp-142 of SPE-C. Location 56 can be occupied by a polaramino acid, preferably Tyr-139 of SPE-C. Location 57 can be occupied bya charged amino acid, preferably Lys-138 of SPE-C. Location 58 can beoccupied by a positively charged amino acid, preferably Lys-135 ofSPE-C. Location 59 can be occupied by a charged amino acid, preferablyGlu-131 of SPE-C. Location 60 can be occupied by a neutral or polaramino acid, preferably Thr-128 of SPE-C.

[0057] Table 2 lists residues of SEB that interact with class II MHC inthe crystal structure of the complex of these two proteins.Superposition of the structures of SEC-3, SEA and TSST-1 with thestructure of the SEB:MHC-II complex indicates amino acids on theseproteins that correspond to the listed SEB residues that interact withMHC-II. Preferred SPE-C mutants have an amino acid substitution at anSPE-C residue that corresponds to a residue in SEB, SEC-3, SEA or TSST-1that interacts with MHC-II. These preferred SPE-C residues include theSPE-A residues listed in Table 2. Corresponding residues from thedifferent proteins are listed across the rows of the table.

[0058] Table 3 lists residues of SEC-3 that interact with the T-cellreceptor in the crystal structure of the complex of these two proteins.Superposition of the structures of SEB, SEA and TSST-1 with thestructure of the SEC-3:T-cell receptor complex indicates amino acids onthese proteins that correspond to the SEC residues that interact withT-cell receptor. Preferred SPE-C mutants have an amino acid substitutionat an SPE-C residue that corresponds to a residue in SEB, SEC-3, SEA orTSST-1 that interacts with the T-cell receptor. These preferred SPE-Cresidues include the SPE-A residues listed in Table 3. Correspondingresidues from the different proteins are listed across the rows of thetable.

[0059] Preferred mutants of SPE-C have amino acid substitutions in atleast one of the locations or for at least one of the amino acidresidues that interacts with the T-cell receptor, MHC-II or the liverrenal tubular cell receptor. These amino acid substitutions can bechosen as described hereinabove to disrupt the interactions. TABLE 1PTSAG CONSERVED RESIDUES TSST-1 SEA SEB SEC-3 SPE-C TYR 13 TYR 30 TYR 28TYR 28 TYR 17 ASP 27 ASP 45 ASP 42 ASP 42 (THR 33) LYS 58 LYS 81 LYS 78LYS 78 (ARG 65) VAL 62 VAL 85 VAL 82 VAL 82 VAL 69 ASP 63 ASP 86 ASP 83ASP 83 ASP 70 GLY 87 GLY 110 GLY 117 GLY 114 GLY 89 THR 89 THR 112 THR119 THR 116 THR 91 LYS 121 LYS 147 LYS 152 LYS 151 LYS 124 LYS 122 LYS148 LYS 153 LYS 152 (ASP 125) LEU 129 LEU 155 LEU 160 LEU 159 (ILE 132)ASP 130 A5P 156 ASP 161 ASP 160 ASP 133 ARG 134 ARG 160 ARG 162 ARG 161ARG 137 LEU 137 LEU 163 LEU 168 LEU 167 LEU 140 LEU 143 LEU 169 LEU 171LEU 170 (ILE 146) TYR 144 TYR 170 TYR 172 TYR 17I TYR 147 GLY 152 GLY182 GLY 185 GLY 184 GLY 156 ASP 167 ASP 197 ASP 199 ASP 199 ASP 171 ILE189 ILE 226 ILE 230 ILE 230 ILE 204

[0060] TABLE 2 RESIDUES INVOLVED IN CLASS II MHC INTERACTIONS SEB TSST-1SEA SEC-3 SPE-C Gln 43 Asn 28 Gln 46 Lys 43 His 34 Phe 44 Ser 29 Phe 47Phe 44 His 35 Leu 45 Leu 48 Leu 45 Leu 36 Tyr 46 Leu 30 Gln 49 Ala 46Asn 37 Phe 47 Gly 31 His 50 His 47 Gln 92 Lys 71 Gln 95 Asn 92 Leu 78Tyr 94 Gln 73 Ala 97 Tyr 94 Ser 80 Ser 96 Gly 99 Ser 96 Met 215 Asn 175Arg 211 Met 215 Ala 186

[0061] TABLE 3 RESIDUES INVOLVED IN TCR INTERACTIONS TSST-1 SEC-3 SEASEB SPE-C ASN 5 GLY 19 THR 21 GLY 19 ASN 8 THR 20 ALA 22 LEV 20 SER 11ASP 8 ASN 23 ASN 25 ASN 23 ASP 12 ASP 11 TYR 26 GLN 28 VAL 26 TYR 15 ASN60 TYR 49 LYS 70 TYR 90 GLY 93 TYR 90 ILE 77 VAL 91 TYR 94 TYR 91 LEU 78GLY 102 ASN 79 LYS 103 VAL 104 SER 106 LYS 103 ARG 145 PHE 176 ASN 171TYR 175 ASP 148 GLN 210 SER 206 GLN 210 ARG 181

[0062] Once a mutant SPE-C toxin is generated having at least one aminoacid change compared with a protein substantially corresponding to thewild type SPE-C toxin, the mutant SPE-C toxin is screened fornonlethality. It is preferred that mutant SPE-C toxins selected fromthis screening are substantially nonlethal in rabbits when administeredusing a miniosmotic pump (as described in Example 4) at the same dose ora greater dose than a wild type SPE-C toxin. A mutant SPE-C toxin orfragment thereof is substantially nonlethal if when administered to arabbit at the same dose as the wild type toxin less than about 10-20% ofrabbits die. Nonlethal mutant toxins are useful in vaccine andpharmaceutical compositions. While not meant to limit the invention, itis believed that some amino acid residues or domains that affectsystemic lethality are separable from other biological activitiesespecially T cell mitogenicity.

[0063] For mutant toxins useful in vaccine compositions it is furtherpreferred that the mutant SPE-C toxins are screened for those that canstimulate an antibody response that neutralizes wild type SPE-C toxinactivity. A method for selecting mutant toxins that can stimulate anantibody response that neutralizes wild type SPE-C toxin activity is todetermine whether the mutant toxin immunoreacts with polyclonalneutralizing antibodies to wild type SPE-C such as available from ToxinTechnologies, Boca Raton, Fla. or Dr. Schlievert. Methods of determiningwhether mutant SPE-C toxins immunoreact with antibodies to wild typeSPE-C toxin include ELISA, Western Blot, Double Immunodiffusion Assayand the like.

[0064] Optionally, the mutant toxins can also be screened to determineif the proteolytic profile of the mutant toxin is the same as the wildtype SPE-C toxin. In some cases, it is preferred that the mutantsgenerated do not substantially change the overall three-dimensionalconformation of the mutant toxin compared with the wild type SPE-Ctoxin. One way of examining whether there has been a change in overallconformation is to look at immunoreactivity of antibodies to wild typeSPE-C toxin and/or to examine the proteolytic profile of mutant SPE-Ctoxins. The proteolytic profile can be determined using such enzymes astrypsin, chymotrypsin, papain, pepsin, subtilisin and V8 protease inmethods known to those of skill in the art. The proteolytic profile ofwild type SPE-C with the sequence shown in FIG. 3 is known. The mutantsthat have a similar profile to that of wild type SPE-C are selected.

[0065] Optionally, mutant toxins can also be screened and selected tohave other changes in biological activity. As described previously,there are several biological activities associated with wild type SPE-Ctoxin. Those biological activities include: 1) fever; 2) STSS; 4)enhancement of endotoxin shock; 5) capillary leak and hypotension; 6)inducing release of cytokines such as IFN gamma, IL-1, TNF-α and TNF-β;7) binding to endothelial cells; 8) binding to MHC class II molecules;9) binding to T-cell receptors; and 10) T-cell mitogenicity(superantigenicity). These biological activities can be measured usingmethods known to those of skill in the art.

[0066] For mutant SPE-C toxins or fragments thereof useful in vaccinecompositions, it is preferred that they are substantially nontoxic andimmunoreactive with neutralizing antibodies to wild type SPE-C.Neutralizing antibodies include those that inhibit the lethality of thewild type toxin when tested in animals. Optionally, mutant SPE-C toxinscan have a change in one or more other biological activities of wildtype SPE-C toxin as described previously.

[0067] Optionally, preferred mutant toxins for vaccine compositions arefurther screened and selected for a lack of potentiation of endotoxinshock. The preferred assay for examining a lack of enhancement ofendotoxin shock is described in Example 3. Rabbits preferably have nodemonstrable bacterial or viral infection before testing. A lack ofpotentiation of or substantially no enhancement of endotoxin shock isseen when less than about 25% of the animals develop shock when themutant SPE-C toxin is coadministered with endotoxin as compared to wildtype SPE-C activity at the same dose. More preferably, none of theanimals develop shock.

[0068] Optionally, preferred mutants for vaccine compositions also arefurther screened and selected for a change in T cell mitogenicity. Achange in T-cell mitogenicity can be detected by measuring T-cellproliferation in a standard 3H thymidine assay using rabbit lymphocytesas described in Example 3; by measuring levels of production ofcytokines such as IFN gamma or TNF-β; by determining the Vβ type of Tcell response or by determining the interaction of the molecules withMHC class II receptors. The preferred method for detecting a decrease inT-cell mitogenicity is to measure T-cell proliferation of rabbitlymphocytes in the presence and absence of the mutant toxin. Responsesof T cells to wild type SPE-C toxin is greatly enhanced above a normalin vitro response to an antigen. A substantial decrease in T cellmitogenicity is seen when the mutant SPE-C toxin does not stimulate a Tcell proliferative response greater than the stimulation with an antigenor negative control. Preferably, a decrease is seen such that the T cellproliferation response to the mutant SPE-C toxin is no more thantwo-fold above background when measured using rabbit lymphocytes at thesame dose as the wild type SPE-C toxin.

[0069] Optionally, the mutant SPE-C toxins useful in vaccinecompositions are further screened and selected for a decrease incapillary leak in endothelial cells. The preferred method is usingporcine aortic endothelial cells as described by Lee et el., J. Infect.Dis. 164:711 (1991). A decrease in capillary leak in the presence ofmutant SPE-C toxins can be determined by measuring a decrease in releaseof a radioactively labeled compound or by a change in the transport of aradioactively labeled compound. A decrease in capillary leak is seenwhen the release or transport of a radioactively labeled compound isdecreased to less than about two fold above background when comparedwith the activity of a wild type toxin.

[0070] The especially preferred mutant SPE-C toxins useful in vaccinecompositions are not biologically active compared with proteins thathave wild type SPE-C toxin activity. By nonbiologically active, it ismeant that the mutant toxin has little or no systemic lethality, haslittle or no enhancement of endotoxin shock and little or no T cellmitogenicity. Preferably, the mutant SPE-C toxins selected for vaccinecompositions substantially lack these biological activities, i.e., theyreact like a negative control or they stimulate a reaction no more thantwo-fold above background.

[0071] Changes in other biological activities can be detected asfollows. Binding to MHC class II molecules can be detected using suchmethods as described by Jardetzky, Nature 368:711 (1994). Changes infever can be detected by monitoring temperatures over time afteradministration of the mutant SPE-C toxins. Changes in the levels ofcytokine production in the presence of mutant SPE-C toxins can bemeasured using methods such as are commercially available and aredescribed by current protocols in immunology. (Ed. Coligan, Kruisbeck,Margulies, Shevach, and Stroker. National Institutes of Health, JohnWiley and Sons, Inc.)

[0072] The especially preferred mutants for vaccine compositions aremutant SPE-C toxins that immunoreact with polyclonal neutralizingantibodies to wild type SPE-C toxin, are nontoxic, and optionally have adecrease in potentiation of endotoxin shock and a decrease in T-cellmitogenicity.

[0073] Advantageously, mutant SPE-C toxins useful in treatment methodscan be generated that have more than one change in the amino acidsequence. It would be desirable to have changes at more than onelocation to minimize any chance of reversion to a molecule havingtoxicity or lethality. For vaccine compositions, it is desirable that amutant toxin with multiple changes can still generate a protectiveimmune response against wild type SPE-C and/or immunoreact withneutralizing polyclonal antibodies to wild type SPE-C. Forpharmaceutical compositions, it is preferred that mutants with multiplechanges are substantially nonlethal while maintaining mitogenicity for Tcells. It is especially preferable to have about 2 to 6 changes. Triplemutants are also contemplated in this application.

[0074] Mutant toxins of SPE-C are useful to form vaccine compositions.The preferred mutants for vaccine compositions have at least one aminoacid change, are nontoxic systemically, and immunoreact with polyclonalneutralizing antibodies to wild type SPE-C.

[0075] Mutant toxins are combined with a physiologically acceptablecarrier. Physiologically acceptable diluents include physiologicalsaline solutions, and buffered saline solutions at neutral pH such asphosphate buffered saline. Other types of physiological carriers includeliposomes or polymers and the like. Optionally, the mutant toxin can becombined with an adjuvant such as Freund's incomplete adjuvant, Freund'sComplete adjuvant, alum, monophosphoryl lipid A, alum phosphate orhydroxide, QS-21 and the like. Optionally, the mutant toxins orfragments thereof can be combined with immunomodulators such asinterleukins, interferons and the like. Many vaccine formulations areknown to those of skill in the art.

[0076] The mutant SPE-C toxin or fragment thereof is added to a vaccineformulation in an amount effective to stimulate a protective immuneresponse in an animal to at least one biological activity of wild typeSPE-C toxin. Generation of a protective immune response can be measuredby the development of antibodies, preferably antibodies that neutralizethe wild type SPE-C toxin. Neutralization of wild type SPE-C toxin canbe measured including by inhibition of lethality due to wild type SPE-Cin animals. In addition, a protective immune response can be detected bymeasuring a decrease in at least one biological activity of wild typeSPE-C toxins such as amelioration or elimination of the symptoms ofenhancement of endotoxin shock or STSS. The amounts of the mutant toxinthat can form a protective immune response are about 0.1 μg to 100 mgper kg of body weight more preferably about 1 μg to about 100 μg/kg bodyweight. About 25 μg/kg of body weight of wild type SPE-C toxin iseffective to induce protective immunity in rabbits.

[0077] The vaccine compositions are administered to animals such asrabbits, rodents, horses, and humans. The preferred animal is a human.

[0078] The mutant SPE-C toxins are also useful to form pharmaceuticalcompositions. The pharmaceutical compositions are useful in therapeuticsituations where a stimulation of T-cell proliferation may be desirable.The preferred mutant SPE-C toxins are those that are nonlethal whilemaintaining T-cell mitogenicity comparable to wild type SPE-C toxin.

[0079] A pharmaceutical composition is formed by combining a mutantSPE-C toxin with a physiologically acceptable carrier such asphysiological saline, buffered saline solutions at neutral pH such asphosphate buffered saline. The mutant SPE-C toxin is combined in anamount effective to stimulate T-cell proliferation comparable to wildtype SPE-C toxin at the same dose. An enhancement in T-cellresponsiveness can be measured using standard 3H thymidine assays withrabbit lymphocytes as well as by measuring T-cell populations in vivousing fluorescence activated T-cell sorters or an assay such as anELISPOT. The range of effective amounts are 100 ng to 100 mg per kg ofbody weight, more preferably 1 μg to 1 mg/kg body weight. For example,these mutant SPE-C toxins could be used either alone or in conjunctionwith interleukin or interferon therapy.

[0080] The invention also includes fragments of SPE-C toxins andfragments of mutant SPE-C toxins. For vaccine compositions, fragmentsare preferably large enough to stimulate a protective immune response. Aminimum size for a B cell epitope is about 4-7 amino acids and for a Tcell epitope about 8-12 amino acids. The total size of wild type SPE-Cis about 235 amino acids including the leader sequence. Fragments arepeptides that are about 4 to 200 amino acids, more preferably about10-50 amino acids.

[0081] Fragments can be a single peptide or include peptides fromdifferent locations joined together. Preferably, fragments include oneor more of the domains as identified in FIG. 1 and as described herein.It is also preferred that the fragments from mutant SPE-C toxins have atleast one change in amino acid sequence and more preferably 1-6 changesin amino acid sequence when compared to a protein substantiallycorresponding to a wild type SPE-C toxin.

[0082] Preferably, fragments are substantially nonlethal systemically.Fragments are screened and selected to have little or no toxicity inrabbits using the miniosmotic pump model at the same or greater dosagethan a protein having wild type SPE-C toxin activity as describedpreviously. It is also preferred that the fragment is nontoxic in humanswhen given a dose comparable to that of a wild type SPE-C toxin.

[0083] For vaccine compositions, it is preferred that the fragmentsinclude residues from the central α helix and/or the N-terminal α helix.For vaccine compositions, it is preferable that a fragment stimulate aneutralizing antibody response to a protein having wild type SPE-C toxinactivity. A fragment can be screened and selected for immunoreactivitywith polyclonal neutralizing antibodies to a wild type SPE-C toxin. Thefragments can also be used to immunize animals and the antibodies formedtested for neutralization of wild type SPE-C toxin.

[0084] For vaccine compositions, especially preferred fragments arefurther selected and screened to be nonbiologically active. Bynonbiologically active, it is meant that the fragment is nonlethalsystemically, induces little or no enhancement of endotoxin shock, andinduces little or no T cell stimulation. Optionally, the fragment can bescreened and selected to have a decrease in capillary leak effect onporcine endothelial cells.

[0085] The fragments screened and selected for vaccine compositions canbe combined into vaccine formulations and utilized as describedpreviously. Optionally, fragments can be attached to carrier moleculessuch as bovine serum albumin, human serum albumin, keyhole limpethemocyanin, tetanus toxoid and the like.

[0086] For pharmaceutical compositions, it is preferred that thefragments include amino acid residues in the N-terminal Domain B βstrands alone or in combination with the central α helix.

[0087] For pharmaceutical compositions, it is preferred that thefragments are screened and selected for nonlethality systemically, andoptionally for little or no enhancement of endotoxin shock as describedpreviously. It is preferred that the fragments retain T cellmitogenicity similar to the wild type SPE-C toxin. Fragments of a mutanttoxin SPE-C can form pharmaceutical compositions as describedpreviously.

[0088] Fragments of mutant SPE-C toxin can be prepared using PCR,restriction enzyme digestion and/or ligation, in vitro mutagenesis andchemical synthesis. For smaller fragments chemical synthesis may bedesirable.

[0089] The fragments of mutant SPE-C toxins can be utilized in the samecompositions and methods as described for mutant SPE-C toxins.

[0090] B. Methods for Using Mutant SPE-C Toxins, Vaccines Compositionsor Pharmaceutical Compositions.

[0091] The mutant SPE-C toxins and/or fragments thereof are useful inmethods for protecting animals against the effects of wild type SPE-Ctoxins, ameliorating or treating animals with STSS, inducing enhancedT-cell proliferation and responsiveness, and treating or amelioratingthe symptoms of guttate psoriasis, rheumatic fever, or invasivestreptococcal infections.

[0092] A method for protecting animals against at least one biologicalactivity of wild type SPE-C toxin involves the step of administering avaccine composition to an animal to establish a protective immuneresponse against at least one biological activity of SPE-C toxin. It ispreferred that the protective immune response is neutralizing andprotects against lethality or symptoms of STSS. The vaccine compositionpreferably includes a mutant SPE-C toxin or fragment thereof that has atleast one amino acid change, that immunoreacts with polyclonalneutralizing antibodies to wild type SPE-C, and is nonlethal.

[0093] The vaccine composition can be administered to an animal in avariety of ways including subcutaneously, intramuscularly,intravenously, intradernally, orally, intranasally, ocularly,intraperitoneally and the like. The preferred route of administration isintramuscularly.

[0094] The vaccine compositions can be administered to a variety ofanimals including rabbits, rodents, horses and humans. The preferredanimal is a human.

[0095] The vaccine composition can be administered in a single ormultiple doses until protective immunity against at least one of thebiological activities of wild type SPE-C is established. Protectiveimmunity can be detected by measuring the presence of neutralizingantibodies to the wild type SPE-C using standard methods. An effectiveamount is administered to establish protective immunity without causingsubstantial toxicity.

[0096] A mutant SPE-C toxin or fragment thereof is also useful togenerate neutralizing antibodies that immunoreact with the mutant SPE-Ctoxin and the wild type SPE-C toxin. These antibodies could be used as apassive immune serum to treat or ameliorate the symptoms in thosepatients that have the symptoms of STSS. A vaccine composition asdescribed above could be administered to an animal such as a horse or ahuman until a neutralizing antibody response to wild type SPE-C isgenerated. These neutralizing antibodies can then be harvested,purified, and utilized to treat patients exhibiting symptoms of STSS.Neutralizing antibodies to wild type SPE-C toxin can also be formedusing wild type SPE-C. However, wild type SPE-C must be administered ata dose much lower than that which induces toxicity such as 1/50 to 1/100of the LD50 of wild type SPE-C in rabbits.

[0097] The neutralizing antibodies are administered to patientsexhibiting symptoms of STSS such as fever, hypotension, group Astreptococcal infection, myositis, fascitis, and liver damage in anamount effective to neutralize the effect of SPE-C toxin. Theneutralizing antibodies can be administered intravenously,intramuscularly, intradermally, subcutaneously, and the like. Thepreferred route is intravenously or for localized infection, topicallyat the site of tissue damage with debridement. It is also preferred thatthe neutralizing antibody be administered in conjunction with antibiotictherapy. The neutralizing antibody can be administered until a decreasein shock or tissue damage is obtained in a single or multiple dose. Thepreferred amount of neutralizing antibodies typically administered isabout 1 mg to 1000 mg/kg, more preferably about 50-200 mg/kg of bodyweight.

[0098] C. DNA Expression Cassettes Encoding Mutant SPE-C Toxins andMethods of Preparation of Such DNA Expression Cassettes

[0099] The invention also includes DNA sequences and expressioncassettes useful in expression of mutant SPE-C toxins and/or fragmentsthereof. An expression cassette includes a DNA sequence encoding amutant SPE-C toxin and/ or fragment thereof with at least one amino acidchange and at least one change in biological function compared to aprotein substantially corresponding to a wild type SPE-C toxin operablylinked to a promoter functional in a host cell. Expression cassettes areincorporated into transformation vectors and mutant SPE-C toxins areproduced in transformed cells. The mutant toxins can then be purifiedfrom host cells or host cell supernatants. Transformed host cells arealso useful as vaccine compositions.

[0100] Mutant SPE-C toxins or fragments thereof can also be formed byscreening and selecting for spontaneous mutants in a similar manner asdescribed for site specific or random mutagenesis. Mutant SPE-C toxinscan be generated using in vitro mutagenesis or semisynthetically fromfragments produced by any procedure. Finally, mutant SPE-C toxins can begenerated using chemical synthesis.

[0101] A method of producing the mutant SPE-C toxins or fragmentsthereof which includes transforming or transfecting a host cell with avector including such an expression cassette and culturing the host cellunder conditions which permit expression of such mutant SPE-C toxins orfragments by the host cell.

[0102] DNA Sequences Encoding Mutant SPE-C Toxins

[0103] A mutant DNA sequence encoding a mutant SPE-C toxin that has atleast one change in amino acid sequence can be formed by a variety ofmethods depending on the type of change selected. A DNA sequenceencoding a protein substantially corresponding to wild type SPE-C toxinfunctions as template DNA used to generate DNA sequences encoding mutantSPE-C toxins. A DNA sequence encoding wild type SPE-C toxin is shown inFIG. 1.

[0104] To make a specific change or changes at a specific location orlocations it is preferred that PCR is utilized according to method ofPerrin et al., cited supra. To target a change to a particular location,internal primers including the altered nucleotides coding for the aminoacid change are included in a mixture also including a 5′ and 3′flanking primers. A 5′ flanking primer is homologous to or hybridizes toa DNA region upstream of the translation start site of the codingsequence for wild type SPE-C. Preferably, the 5′ flanking region isupstream of the speA promoter and regulatory region. For example, a 5′flanking primer can be homologous to or hybridize to a region about 760bases upstream of the translation start site. A downstream flankingprimer is homologous to or hybridizes to a region of DNA downstream ofthe stop codon of the coding sequence for wild type SPE-C. It ispreferred that the downstream flanking primer provides fortranscriptional and translational termination signals. For example, a 3′flanking primer can hybridize or be homologous to a region 200 basepairs downstream of the stop codon for the coding sequence of SPE-C. Theupstream and downstream flanking primers are present in every PCRreaction to ensure that the resulting PCR product includes the speCpromoter and upstream regulatory region and transcriptional andtranslation termination signals. Other upstream and downstream primerscan readily be constructed by one of skill in the art. While preferred,it is not absolutely necessary that the native speC promoter andupstream regulatory region be included in the PCR product.

[0105] Internal primers can be designed to generate a change at aspecific location utilizing a DNA sequence encoding wild type SPE-C.Primers can be designed to encode a specific amino acid substitution ata specific location. Primers can be designed to result in randomsubstitution at a particular site as described by Rennell et al., J.Mol. Biol. 22:67 (1991). Primers can be designed that result in adeletion of an amino acid at a particular site. Primers can also bedesigned to add coding sequence for an additional amino acid at aparticular location.

[0106] Primers are preferably about 15 to 50 nucleotides long, morepreferably 15 to 30 nucleotides long. Primers are preferably prepared byautomated synthesis. The 5′ and 3′ flanking primers preferably hybridizeto the flanking DNA sequences encoding the coding sequence for the wildtype SPE-C toxin. These flanking primers preferably include about 10nucleotides that are 100% homologous or complementary to the flankingDNA sequences. Internal primers are not 100% complementary to DNAsequence coding for the amino acids at location because they encode achange at that location. An internal primer can have about 1 to 4mismatches from the wild type SPE-C sequence in a primer about 15 to 30nucleotides long. Both flanking primers and internal primers can alsoinclude additional nucleotides that encode for restriction sites andclamp sites, preferably near the end of the primer. Hybridizationconditions can be modified to take into account the number of mismatchespresent in the primer in accord with known principles as described bySambrook et al. Molecular Cloning-A laboratory manual, Cold SpringHarbor Laboratory Press, (1989).

[0107] More than one internal primer can be utilized if changes at morethan one site are desired. A PCR method for generating site-specificchanges at more than one location is described in Aiyar et al. citedsupra. Another method is described in Example 5.

[0108] In one method, a DNA sequence encoding a mutant SPE-C toxin withone change at a particular site is generated and is then used as thetemplate to generate a mutant DNA sequence with a change at a secondsite. In the first round of PCR, a first internal primer is used togenerate the mutant DNA sequence with the first change. The mutant DNAsequence with the first change is then used as the template DNA and asecond internal primer coding for a change at a different site is usedto form a DNA sequence encoding a mutant toxin with changes in aminoacid sequences at two locations. PCR methods can be utilized to generateDNA sequences with encoding amino acid sequences with about 2 to 6changes.

[0109] A preferred PCR method is as described by Perrin et al. citedsupra. Briefly, the PCR reaction conditions are: PCR is performed in a100 ul reaction mixture containing 10 mM Tris-HCl (pH=8.3), 50 mM KCl,1.5 mM MgCl2, 200 uM each dNTP, 2 ng template plasmid DNA, 100 pmolesflanking primer, 5 pmoles internal primer, and 2.5 units of Ampli TaqDNA polymerase (Perkin Elmer Cetus). In the second amplification step,the composition of the reaction mix is as above except for equalmolarity (5 pmoles each) of flanking primer and megaprimer and 1 ugtemplate. PCR is conducted for 30 cycles of denaturation at 94° C.×1minute, annealing at 37° C. or 44° C.×2minutes and elongation at 72° C.for 3 minutes.

[0110] The PCR products are isolated and then cloned into a shuttlevector (such as pMIN 164 as constructed by the method of Murray et al,J. Immunology 152:87 (1994) and available from Dr. Schlievert,University of Minnesota, Mpls, Minn.). This vector is a chimera of E.coli plasmid pBR328 which carries ampicillin resistance and thestaphylococcal plasmid pE194 which confers erythromycin resistance. Theligated plasmid mixtures are screened in E. coli for toxin productionusing polylconal neutralizing antibodies to wild type SPE-C from ToxinTechnologies, Boca Raton, Fla. or from Dr. Schlievert. The mutant SPE-Ctoxins are sequenced by the method of Hsiao et al., Nucleic Acid Res.19:2787 (1991) to confirm the presence of the desired mutation andabsence of other mutations.

[0111] It will be understood by those of skill in the art that due tothe degeneracy of the genetic code a number of DNA sequences can encodethe same changes in amino acids. The invention includes DNA sequenceshaving different nucleotide sequences but that code for the same changein amino acid sequence.

[0112] For random mutagenesis at a particular site a series of primersare designed that result in substitution of each of the other 19 aminoacids or a non-naturally occurring amino acid or analog at a particularsite. PCR is conducted in a similar manner as described above or by themethod described by Rennell et al., cited supra. PCR products aresubcloned and then toxin production can be monitored by immunoreactivitywith polylconal neutralizing antibodies to wild type SPE-C. The presenceof a change in amino acid sequence can be verified by sequencing of theDNA sequence encoding the mutant SPE-C toxin. Preferably, mutant toxinsare screened and selected for nonlethality.

[0113] Other methods of mutagenesis can also be employed to generaterandom mutations in the DNA sequence encoding the wild type SPE-C toxin.Random mutations or random mutagenesis as used in this context meansmutations are not at a selected site and/or are not a selected change. Abacterial host cell including a DNA sequence encoding the wild typeSPE-C toxin can be mutagenized using other standard methods such aschemical mutagenesis, and UV irradiation. Mutants generated in thismanner can be screened for toxin production using polyclonalneutralizing antibodies to wild type SPE-C. However, further screeningis necessary to identify mutant toxins that have at least one change ina biological activity, preferably that are nonlethal. Spontaneouslyarising mutants can also be screened for at least one change in abiological activity from wild type SPE-C.

[0114] Random mutagenesis can also be conducted using in vitromutagenesis as described by Anthony-Cahill et al., Trends Biochem. Sci.14: 400 (1989).

[0115] In addition, mutant SPE-C toxins can be formed using chemicalsynthesis. A method of synthesizing a protein chemically is described inWallace, FASEB J. 7:505 (1993). Parts of the protein can be synthesizedand then joined together using enzymes or direct chemical condensation.Using chemical synthesis would be especially useful to allow one ofskill in the art to insert non-naturally occurring amino acids atdesired locations. In addition, chemical synthesis would be especiallyuseful for making fragments of mutant SPE-C toxins.

[0116] Any of the methods described herein would be useful to formfragments of mutant SPE-C toxins. In addition, fragments could bereadily generated using restriction enzyme digestion and/or ligation.The preferred method for generating fragments is through direct chemicalsynthesis for fragment of 20 amino acids or less or through geneticcloning for larger fragments.

[0117] DNA sequences encoding mutant toxins, whether site-specific orrandom, can be further screened for other changes in biological activityfrom wild type SPE-C toxin. The methods for screening for a change in atleast one biological activity are described previously. Once selectedDNA sequences encoding mutant SPE-C toxins are selected for at least onechange in biological activity, they are utilized to form an expressioncassette.

[0118] Formation of an expression cassette involves combining the DNAsequences coding for mutant SPE-C toxin with a promoter that providesfor expression of a mutant SPE-C toxin in a host cell. For those mutantSPE-C toxins produced using PCR as described herein, the native speCpromoter is present and provides for expression in a host cell.

[0119] Optionally, the DNA sequence can be combined with a differentpromoter to provide for expression in a particular type of host cell orto enhance the level of expression in a host cell. Preferably, thepromoter provides for a level of expression of the mutant SPE-C toxin sothat it can be detected with antibodies to SPE-C. Other promoters thatcan be utilized in prokaryotic cells include PLAC, PTAC, T7, and thelike.

[0120] Once the DNA sequence encoding the mutant SPE-C toxin is combinedwith a suitable promoter to form an expression cassette, the expressioncassette is subcloned into a suitable transformation vector. Suitabletransformation vectors include at least one selectable marker gene andpreferably are shuttle vectors that can be amplified in E. coli and grampositive microorganisms. Examples of suitable shuttle vectors includepMIN 164, and pCE 104. Other types of vectors include viral vectors suchas the baculovirus vector, SV40, poxviruses such as vaccinia, adenovirusand cytomegalovirus. The preferred vector is a pMIN 164 vector, ashuttle vector that can be amplified in E. coli and S. aureus.

[0121] Once a transformation vector is formed carrying an expressioncassette coding for a mutant SPE-C toxin, it is introduced into asuitable host cell that provides for expression of the mutant SPE-Ctoxin. Suitable host cells are cells that provide for high level ofexpression of the mutant toxin while minimizing the possibility ofcontamination with other undesirable molecules such as endotoxin andM-proteins. Suitable host cells include mammalian cells, bacterial cellssuch as S. aureus, E. coli and Salmonella spp., yeast cells, and insectcells.

[0122] Transformation methods are known to those of skill in the art andinclude protoplast transformation, liposome mediated transformation,calcium phosphate precipitation and electroporation. The preferredmethod is protoplast transformation.

[0123] Transformed cells are useful to produce large amounts of mutantSPE-C toxin that can be utilized in vaccine compositions. A transformedmicroorganism can be utilized in a live, attenuated, or heat killedvaccine. A transformed microorganism includes mutant toxin SPE-C inamounts sufficient to stimulate a protective immune response to wildtype SPE-C. Preferably, the mutant SPE-C toxin is secreted. Themicroorganism is preferably nonpathogenic to humans and includes amutant toxin with multiple amino acid changes to minimize reversion to atoxic form. The microorganism would be administered either as a live orheat killed vaccine in accordance with known principles. Preferredmicroorganisms for live vaccines are transformed cells such asSalmonella spp.

[0124] A viral vector including an expression cassette with a DNAsequence encoding a mutant SPE-C toxin or fragment thereof operablylinked to a promoter functional in a host cell can also be utilized in avaccine composition as described herein. Preferably, the promoter isfunctional in a mammalian cell. An example of a suitable viral vectorincludes pox viruses such as vaccinia virus, adenoviruses,cytomegaloviruses and the like. Vaccinia virus vectors could be utilizedto immunize humans against at least one biological activity of a wildtype SPE-C toxin.

[0125] The invention also includes a vaccine composition comprising annucleic acid sequence encoding a mutant SPE-C toxin or fragment thereofoperably linked to a promoter functional in a host cell. The promoter ispreferably functional in a mammalian host cell. The nucleic acidsequence can be DNA or RNA. The vaccine composition is delivered to ahost cell or individual for expression of the mutant SPE C toxin orfragment thereof within the individuals own cells. Expression of nucleicacid sequences of the mutant SPE C toxin or fragment thereof in theindividual provides for a protective immune response against the wildtype SPE C toxin. Optionally, the expression cassette can beincorporated into a vector. A nucleic acid molecule can be administeredeither directly or in a viral vector. The vaccine composition can alsooptionally include a delivery agent that provides for delivery of thevaccine intracellularly such as liposomes and the like. The vaccinecomposition can also optionally include adjuvants or otherimmunomodulatory compounds, and additional compounds that enhance theuptake of nucleic acids into cells. The vaccine composition can beadministered by a variety of routes including parenteral routes such asintravenously, intraperitoneally, or by contact with mucosal surfaces.

[0126] Conditions for large scale growth and production of mutant SPE-Ctoxin are known to those of skill in the art. A method for purificationof mutant SPE-C toxins from microbial sources is as follows. S. aureuscarrying the mutant or the wild type speCs in pMIN64 are grown at 37° C.with aeration to stationary phase in dialyzable beef heart medium,containing 5 μg/ml of erythromycin. Cultures are precipitated with fourvolumes of ethanol and proteins resolubilized in pyrogen free water. Thecrude preparations are subjected to successive flat bed isoelectricfocusing separations in pH gradients of 3.5 to 10 and 4 to 6. Thefractions that are positive for toxin by antibody reactivity areextensively dialyzed against pyrogen free water, and an aliquot of eachis tested for purity by SDS polyacrylamide gel electrophoresis in 15%(weight/volume) gels. Polyclonal neutralizing antibodies to SPE-C areavailable from Toxin Technologies, Boca Raton, Fla. or Dr. Schlievert.Other methods of purification including column chromatography or HPLCcan be utilized.

[0127] This invention can be better understood by way of the followingexamples which are representative of the preferred embodiments thereof,but which are not to be construed as limiting the scope of theinvention.

EXAMPLE 1 Cloning and Expression of SPE-C Wild Type

[0128] Cloning and Expression of speC in E. coi

[0129] To obviate the need of toxin detection for gene isolation,oligonucleotides specific for the SPE-C gene were synthesized and usedto screen a streptococcal genomic library. Purified streptococcal DNAfrom strain T18P was partially digested with the restrictionendonuclease Sau 3A and separated on 0.7% agarose gel. Fragments in the4-8 kilobase range were eluted from the gel and ligated to vectorplasmid pBR328, which had been linearized with BAM H1 anddephosphorylated to prevent self-ligation. The ligated DNA was then usedto transform competent E. coli RR1 cells to ampicillin resistance.Transformants were grown on nitrocellulose filters overlayed on LB agarcontaining ampicillin. Replica filters were prepared, and approximately1500 recombinant colonies were screened for the presence of the speCgene by colony hybridization to radiolabeled synthetic oligonucleotides.Two families of mixed sequence oligonucleotides were derived from thehexapeptide sequence, Asp-Ser-Lys-Lys-Asp-Ile, which corresponds to thefirst six amino acids of the amino terminus of the mature SPE-C protein(FIG. 1). The oligonucleotides were split into two families to controlthe redundancy of the probes and thereby minimize nonspecifichybridization. Two colonies were found to hybridize with oligonucleotidefamily A. Colonies hybridizing to family B were not found. Thehybridizing clones were assayed for SPE-C expression by precipitationwith SPE-C antiserum in Ouchterlony immunodiffusion tests. The lysatefrom one of the selected clones formed a precipitin line of identitywith purified SPE-C. The recombinant plasmid containing speC wasdesignated pUMN 501. Culture supernatant fluid from RRlpUMN 501 wasfound not to contain detectable amounts of SPE-C, suggesting that E.coli was unable to secrete the toxin.

[0130] Subcloning

[0131] The insert within pUMN 501 was approximately 4.0 kilobases andbordered by Sau 3A sites (FIG. 2). Digestion with XbaI yielded a 2.4 anda 1.6 kilobase fragment, neither of which directed speC expression whenligated to pUC13 and transformed into E. coli JM101 (pUMN 512 and pUMN511, respectively). The larger Sau 3A-Sal I fragment (3.3 kilobases)expressed speC in E. coli JM101 pUMN 513). The gene was expressed ineither orientation with respect to the plasmid promoter, suggesting thatthe native streptococcal promoter was present within the insert andfunctional in E. coli. The speC gene was further localized by cloning a3.3 kilobase Sau 3A-Sal I fragment into M13 bacteriophage and utilizingthe procedure of Dale et al. Plasmid 13:31-40 (1985) to generatedeletion subclones. A 1.7 kilobase fragment isolated from an M13subclone and ligated to pUC13 (pUMN 521), was capable of expression speCin E. coli.

EXAMPLE 2 Biochemical Characterization of E. coli-derived SPE-C

[0132] SPE-C encoded by UMN 501 was partially purified from extracts ofE. coli RR1 by ethanol precipitation followed by preparative isoelectricfocusing in a pH gradient of 3.5-10. E. coli-derived toxin migrated tothe same approximate location, (between 6.5 and 7.2), as thestreptococcal-derived toxin. E. coli and streptococcal-derived SPE-C hadidentical molecular weights of 24000 in SDS-PAGE. Though additionalproteins were present in the E. coli preparation, only the 24000 mwprotein reacted when tested by an immunoblot technique usingSPE-C-specific antiserum.

EXAMPLE 3 Biological Characterization of E. coli-derived SPE-C

[0133]E. coli and streptococcal-derived SPE-C were compared forlymphocyte mitogenicity. Rabbit splenocytes (2×10⁵ cells) were exposedto approximately 0.01 ug SPE-C from S. pyogenes or E. coli(pUMN 501).After 3 days, the cultures were pulsed with 1 uCi [³H]-thymidine andincubated for 24 h, after which incorporation of radiolabel intocellular DNA was quantified. Both toxin preparations induced a similarmitogenic response. Incubation with SPE-C antiserum significantlyreduced the mitogenic response of both cloned and streptococcal-derivedtoxin.

[0134] Streptococcal and E. coli-derived SPE-C were also compared forpyrogenicity and enhancement of lethal endotoxin shock in rabbits. Thestreptococcal and E. coli-derived SPE-C were equally pyrogenic; theaverage rise in temperature for both preparations was 1.0 C. after 4 h.The fever responses were monophasic, rather than biphasic as ischaracteristic of endotoxin. This suggests that the fever wasattributable to SPE-C and not due to endotoxin contamination. Both E.coli and streptococcal-derived SPE-C treated animals showed enhancedsusceptibility to endotoxin shock. All of the control rabbits receivingonly PBS and endotoxin, survived.

[0135] These studies confirm that SPE-C is expressed in E. coli in abiologically active form, and activities attributed to SPE-C were notdue to a copurified streptococcal contaminant.

EXAMPLE 4 Administration and Immunization of Rabbits with RecombinantlyProduced SPE-C (wt)

[0136] Recombinantly produced SPE-C was administered to rabbits at atotal dose of 200 μg/in 0.2 ml over a 7-day period. The results indicatethat animals treated with SPE-C developed the criteria of STSS withnearly all animals succumbing in the 7-day period (data not shown). Thesymptoms of STSS in rabbits include weight loss, diarrhea, mottled face,fever, red conjunctiva and mucosa, and clear brown urine. As expected,control non-toxin treated animals remained healthy. Two other majorobservations were made: 1) fluid replacement provided completeprotection to the animals as expected, and 2) none of the toxin treatedanimals developed necrotizing fascitis and myositis, indicating factorsother than, or in addition to, SPE-C are required for the soft tissuedamage. Development of the clinical features of STSS correlates withadministration of SPE-C.

EXAMPLE 5 Preparation of Double or Triple Mutants of SPE-C Using PCR

[0137] There are a number of methods that are used to generate double ortriple mutant SPE-C toxins or fragments thereof.

[0138] Mutant SPE-C toxins with two or more changes in amino acidsequences are prepared using PCR as described previously. In a first PCRreaction, a first internal primer coding for the first change at aselected site was combined with 5′and 3′ flanking primers to form afirst PCR product. The first PCR product was a DNA sequence coding for amutant SPE-C toxin having one change in amino acid sequence. This firstPCR product then served as the template DNA to generate a second PCRproduct with two changes in amino acid sequence compared with a proteinhaving wild type SPE-C activity. The first PCR product was the templateDNA combined with a second internal primer coding for a change in aminoacid at a second site. The second internal primer was also combined withthe 5′ and 3′ flanking primers to form a second PCR product. The secondPCR product was a DNA sequence encoding a mutant SPE-C toxin withchanges at two sites in the amino acid sequence. This second PCR productwas then used as a template in a third reaction to form a product DNAsequence encoding a mutant SPE-C toxin with changes at three sites inthe amino acid sequence. This method is utilized to generate DNAsequences encoding mutant toxins having more than one change in theamino acid sequence.

[0139] An alternative method to prepare DNA sequences encoding more thanone change is to prepare fragments of DNA sequence encoding the changeor changes in amino acid sequence by automated synthesis. The fragmentsare then subcloned into the wild type SPE-C coding sequence usingseveral unique restriction sites. Restriction sites are known to thoseof skill of the art and are readily determined from the DNA sequence ofa wild type SPE-C toxin. The cloning is done in a single step with athree fragment ligation method as described by Revi et al. Nucleic AcidRes. 16: 1030 (1988).

EXAMPLE 6 Evaluation of Single and Double Mutants of SPE-C

[0140] Three single amino acid mutants of SPE C were made: a) Y15A inwhich tyrosine at position 15 was changed to alanine, b) Y17A in whichtyrosine at position 17 was changed to alanine, c) N38A in whichasparagine at position 38 was changed to alanine. Two double amino acidmutants of SPE C also were made: a) Y15A/N38A, b) Y17A/N38A. All mutantswere constructed by use of the Quik Change method (Stratagene, La Jolla,Calif.) with the speC containing plasmid pUMN521 as template. pUMN521contains the SPE C gene (speC) in pUC13 (Goshorn et al.).

[0141] The single amino acid mutant proteins were produced inEscherichia coli in 100 ml cultures. After growth in the presence of 50μg/ml ampicillin, the E. coli cultures were treated with 400 ml −20° C.ethanol to lyse cells and precipitate SPE C mutant proteins. pUMN521 inE. coli was treated comparably for use as a positive control. Theprecipitates were collected and restored to 1 ml. Toxin concentrationswere estimated to be 25 μg/ml.

[0142] Wild type SPE C from pUMN521 and the three single amino acidmutants were evaluated for capacity to induce rabbit splenocyteproliferation over a toxin dose range of 0.25 to 2.5×10⁻⁵ or 2.5×10⁶. Asindicated in FIG. 7, the Y15A and N38A mutants were approximately onehalf as mitogenic as the wild type. The Y17A mutant was essentiallynonmitogenic (FIG. 8).

[0143] The double mutants Y15A/N38A and Y17A/N38A were also tested forability to stimulate rabbit splenocytes compared to wild type toxin(FIG. 9). Both mutants stimulated rabbit splenocytes only to one-fourththat seen by comparable amounts of wild type toxin.

[0144] Both double mutants were also tested for capacity to enhanceendotoxin shock. Three rabbits/group were challenged intravenously with5 μg/kg of mutants or wild type toxin. After 4 hours, the same animalswere challenged with 10 μkg/kg Salmonella typhimurium endotoxin (1/50LD₅₀). Deaths were recorded over a 48 hour time period (Table 4). Asindicated, neither double mutant caused lethality in the rabbits. TABLE4 Capacity of double amino acid mutants of SPE C to enhance rabbitsusceptibility to endotoxin shock. Number Dead Treatment Protein{overscore (Total Rabbits tested)} SPE C wild type 3/3 Y15A/N38A 0/3Y17A/N38A 0/3

[0145] One week after challenge of the rabbits used in Table 4, theanimals were euthanized and examined for gross tissue damage. Allorgans, including liver, spleen, kidneys, lungs and heart appearednormal. This is consistent with the lack of toxicity of the doublemutants.

[0146] Three rabbits/group were also immunized with two weekly doses of25 μg of SPE C double mutants emulsified in Freund's incompleteadjuvant. The animals were then rested for 5 days. 0.5 ml of blood wascollected from each animal and pooled for collection of Y15A/N38A andY17A/N38A sera. The sera from these pools was compared to preimmunepooled serum by peroxidase based ELISA (Hudson and Hay reference) forantibodies against purified streptococcal derived wild type SPE A. Table5 summarizes the results of the ELISA. TABLE 5 ELISA antibody titers ofrabbits immunized against Y15A/N38A and Y17A/N38A mutants of SPE C.*Sample tested ELISA titer: Y15A/N38A Preimmune <10* Immune   80Y17A/N38A Preimmune <10 Immune   80

[0147] The immunized animals were then challenged with 5 μg/kg of wildtype SPE C and then 4 hours later 10 μg/kg of Salmonella typhimuriumendotoxin as a test for capacity to immunize against lethality. Table 6indicates the animals were protected from challenge and were thus immuneto SPE C. TABLE 6 Challenge of Y15A/N38A and Y17A/N38A immune animalswith wild type SPE C and endotoxin. Rabbit Group Number Dead/TotalTested Nonimmune 2/2 Y15A/N38A immune 0/3 Y17A/N38A immune 0/3

[0148] Additional single amino acid mutants of SPE C were also prepared.These include residues in the three major domains that may be requiredfor toxicity. These include the T cell receptor binding domain, theclass II MHC binding domain, and residues along the back of the centraldiagonal alpha helix. The residues changed and the effect of themutation on T cell miotgenicity are listed in Table 7. TABLE 7 Effect ofmutants of SPE C on T lymphocyte mitogenicity and lethality BiologicalActivity Mutant Mitogenicity^(a) Lethality^(b) D12A Not tested 0/2 H35A100% of wild type Not tested N38D Not Tested 0/2 K135D 50% of wild typeNot tested K138D 62% of wild type Not tested Y139A 54% of wild type Nottested D142N 52% of wild type Not tested

[0149] The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

[0150] All publications and patent applications in this specificationare indicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

What is claimed is:
 1. A mutant SPE-C toxin or fragment thereof, whereinthe mutant has at least one amino acid change and is substantiallynonlethal compared with a protein substantially corresponding to wildtype SPE-C toxin.
 2. A mutant SPE-C toxin according to claim 1, whereinthe mutant SPE-C toxin comprises one to six amino acid substitutions;and wherein at least one of the substituted amino acids is positioned ina β-barrel of a B-subunit, in an N-terminal alpha helix, in a diagonalalpha helix, or in a surface groove between subunit A and subunit B. 3.A mutant SPE-C toxin according to claim 1, wherein the mutant SPE-Ctoxin comprises one to six amino acid substitutions; and wherein atleast one of the substituted amino acids is aspartic acid-12,tyrosine-15, tyrosine-17, histidine-35, asparagine-38, lysine-135,lysine-138, tyrosine-139, or aspartic acid-142.
 4. The mutant SPE-Ctoxin of claim 3, wherein the at least one amino acid substitutioncomprises the substitution of aspartic acid-12 to alanine, glutamicacid, asparagine, glutamine, lysine, arginine, serine, or threonine; thesubstitution of tyrosine-15 to phenylalanine, alanine, glycine, serine,or threonine; the substitution of tyrosine-17 to phenylalanine, alanine,glycine, glutamic acid, lysine, arginine, aspartic acid, serine, orthreonine; the substitution of histidine-35 to phenylalanine, alanine,glycine, glutamic acid, lysine, arginine, aspartic acid, tyrosine,phenylalanine, serine, or threonine; the substitution of asparagine-38to alanine, aspartic acid, glutamic acid, lysine or arginine; thesubstitution of lysine-135 to glutamic acid or aspartic acid; thesubstitution of lysine-138 to glutamic acid or aspartic acid; thesubstitution of tyrosine-139 to phenylalanine, alanine, glycine,glutamic acid, lysine, arginine, aspartic acid, serine, or threonine; orthe substitution of aspartic acid-142 to alanine, glutamic acid,asparagine, glutamine, serine, threonine, lysine or arginine.
 5. Themutant SPE-C toxin of claim 4, wherein the at least one amino acidsubstitution comprises the substitution of aspartic acid-12 to alanine,the substitution of tyrosine-15 to alanine, the substitution oftyrosine-17 to alanine, the substitution of histidine-35 to alanine, thesubstitution of asparagine-38 to aspartic acid, the substitution oflysine-135 to aspartic acid; the substitution of lysine-138 to asparticacid; the substitution of tyrosine-139 to alanine, or the substitutionof aspartic acid-142 to asparagine.
 6. The mutant SPE-C toxin of claim3, wherein the at least one amino acid substitution comprisessubstitution of tyrosine-15 and asparagine-38.
 7. The mutant SPE-C toxinof claim 6, wherein the substitutions are tyrosine-15 to alanine andasparagine-38 alanine.
 8. The mutant SPE-C toxin of claim 3, wherein theat least one amino acid substitution comprises substitution oftyrosine-17 and asparagine-38.
 9. The mutant SPE-C toxin of claim 8,wherein the substitutions are tyrosine-17 to alanine and asparagine-38alanine.
 10. The mutant SPE-C toxin of claim 1, wherein the mutant hasat least one of the following characteristics: the mutant has a decreasein mitogenicity for T-cells, the mutant does not substantially enhanceendotoxin shock, the mutant is not lethal, or the mutant is nonlethalbut retains mitogenicity comparable to that of the wild type SPE-Ctoxin.
 11. A vaccine for protecting animals against at least onebiological activity of wild-type SPE-C comprising: an effective amountof at least one mutant SPE-C toxin according to claim
 1. 12. Apharmaceutical composition comprising: a mutant SPE-C according to claim1 in admixture with a physiologically acceptable carrier.
 13. A DNAsequence encoding a mutant SPE-C toxin according to claim
 1. 14. Astably transformed host cell comprising a DNA sequence according toclaim
 13. 15. A method for protecting an animal against at least onebiological activity of a wild type SPE-C comprising: administering avaccine according to claim 11 to an animal.
 16. A method for reducingsymptoms associated with toxic shock comprising: administering a vaccineaccording to claim 11 to an animal.